microRNAs (miRNAs) are ~22 nucleotide non-coding RNAs that usually repress genes by imperfectly binding to the 3'UTR of the target mRNA which causes translational repression and mRNA destabilization. miRNA dysregulation has been observed in many cancers and many miRNAs are not only involved in the initiation and progression of cancer but may have therapeutic potential because they target tumor suppressor genes or oncogenes. Bladder cancer, which usually presents as transitional cell carcinoma (TCC), is the fifth most common cancer in the United States. Preliminary results from miRNA profiling indicate that miRNA misexpression has severe consequences in TCC tumorigenesis and some miRNAs may be tumor suppressors or oncogenes. Our study also shows that some silenced tumor suppressor miRNAs can be reactivated by epigenetic treatment. Furthermore, we have developed a flexible platform using the CMV promoter to restore expression of multiple tumor suppressor miRNAs from a single engineered transcript. This novel expression vector can inhibit tumor cell growth by targeting multiple oncogenes or oncogenic pathways. In this proposal, based on our strong preliminary results, we plan to focus on: 1) identifying specific miRNAs for diagnostic and prognostic purposes for bladder cancer patients; 2) reactivating silenced tumor suppressor miRNAs by epigenetic treatment; 3) characterizing the role of miRNAs during tumorigenesis and re-expressing identified tumor suppressor miRNAs in cancer cells with a multiple miRNA expression vector. Completion of these specific aims will provide an important step towards the clinical application of using miRNAs as diagnostic and/or prognostic markers and as therapeutic targets in bladder cancer patients.